CN113125714B - Human immunodeficiency virus antibody detection kit and application thereof - Google Patents
Human immunodeficiency virus antibody detection kit and application thereof Download PDFInfo
- Publication number
- CN113125714B CN113125714B CN202011637955.XA CN202011637955A CN113125714B CN 113125714 B CN113125714 B CN 113125714B CN 202011637955 A CN202011637955 A CN 202011637955A CN 113125714 B CN113125714 B CN 113125714B
- Authority
- CN
- China
- Prior art keywords
- hiv
- kit
- reagent
- sample
- polysaccharide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
- G01N33/56988—HIV or HTLV
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
Abstract
A human immunodeficiency virus antibody detection kit comprising a reagent R1, said reagent R1 comprising a first buffer solution and suspended therein receptor particles capable of generating chemiluminescent signals upon interaction with reactive oxygen species, characterized in that: the receptor particles comprise a carrier, the interior of the carrier is filled with a luminous composition, and the surface of the carrier is bonded with HIV antigen; the receptor particles have a ZETA potential value of not greater than 0mV and not less than-15 mV. The acceptor particle ZETA potential value of the R1 reagent in the kit is not higher than 0mV and not lower than-15 mV; the R1 reagent is more stable, can meet the commercial requirement of mass production, has ultrahigh sensitivity and has a very wide detection range.
Description
Technical Field
The technical scheme relates to the field of chemiluminescence detection, in particular to a human immunodeficiency virus antibody detection kit and application thereof.
Background
Immunoassays have been developed for over half a century. The separation of the test substance from the reaction system in the measurement process may be classified into heterogeneous (heterogenic) immunoassays and Homogeneous (homogenic) immunoassays. Heterogeneous immunoassay refers to the main method in the prior immunoassay, wherein various related reagents are required to be separated after mixing reaction in the operation process of marking a probe, and the detection is performed after separating an object to be detected from a reaction system. Such as enzyme-linked immunosorbent assay (ELISA) and magnetic particle chemiluminescence, which are widely known. Homogeneous immunoassay refers to a method in which an analyte is mixed with a reagent in a reaction system and then directly measured in a measurement process without redundant separation or washing steps. Up to now, various sensitive detection methods are applied to homogeneous immunoassays, such as optical detection methods, electrochemical detection methods, and the like.
Acquired immunodeficiency syndrome (AIDS) is a serious infectious disease that damages the immune function of the human body after the HIV virus, i.e., human immunodeficiency virus (HIV for short), invades the human body, causes various incurable infections and tumors in the human body, and finally causes death of the infected person. The HIV infection is mainly characterized by serious damage to immune system, destruction of T4 lymphocytes, reduced body resistance, induction of serious infection and rare cancer, and patients are prone to various rare diseases and finally die due to long-term consumption and general failure. Thus, the prevention and treatment of acquired immunodeficiency syndrome is the most important link in HIV detection.
At present, the detection method of HIV is as follows:
A. enzyme-linked immunosorbent assay (ELISA)
There are as many as 8 ELISA methods currently used. Their specificity and sensitivity are over 99%.
B. Particle agglomeration method (PA)
PA is a rapid, simple screening method. If the genus is positive, the condition should be confirmed by WB. The PA does not need any special instrument, and the result can be distinguished by naked eyes. The whole process takes only 5 minutes. The disadvantages are false positives and high price.
C. Rapid reagents
a) Human Immunodeficiency Virus (HIV) 1+2 type antibody diagnostic reagent (colloidal selenium method)
The product is only used for the on-site preliminary screening of gratuitous blood donors and clinical emergency, and the positive person is detected and needs to be further screened and confirmed.
b)InstantCHEK TM HIVL+2 gold-labeled rapid diagnostic reagent
InstantCHEK TM HIV1+2 is a rapid, simple, sensitive assay for the detection of antibodies to the HIV-viruses (HIV-1 and HIV-2). The method is suitable for primary screening, and can be used for detecting positive person by the reagent by another method such as ELISA or protein printing method.
HIV-antibody confirmation experiments
Immunoblotting (WB), band immunoassay (LIATEK HIV iii), radioimmunoprecipitation (RIPA) and Immunofluorescence (IFA). The usual confirmation test method in China is WB.
a) Immunoblotting experiment (western blot, WB)
The assay widely used for diagnosis of many infectious diseases is the first assay for confirming HIV antibodies in terms of etiology diagnosis of HIV, and the detection result of WB is often used as a "gold standard" for discriminating the quality of other assays.
WB is generally not less sensitive than the primary screening assay, but its specificity is very high, mainly based on the separation and concentration and purification of the different antigen components of HIV, enabling detection of antibodies directed against the different antigen components, thus enabling discrimination of the accuracy of the primary screening assay by WB methods. From the WB validation test results, it is seen that the preliminary screening test, despite selection of a better quality reagent, such as a third generation ELISA, still has false positives that must be passed through the validation test to give accurate results.
b) Immunofluorescence experiment (IFA)
The IFA method is economical, simple and fast, and has been proposed by the FDA for diagnosis of WB uncertainty samples. However, expensive fluorescence microscopy is required, well-trained technicians are required, the observation and interpretation results are susceptible to subjective factors, and the results are not suitable for long-term storage, and IFA is not suitable for development and use in a general laboratory.
The methods have the difficult problems that the methods are difficult to overcome, and the chemiluminescence method has the advantages of high sensitivity, high specificity, wide linearity, rapidness, few influencing factors, accurate results and the like, and is the disease detection method which is most widely applied in recent years. The method is gradually applied to detection of HIV at present, and is helpful for realizing early discovery, early diagnosis and early treatment of infection, and reducing false positive and omission rate.
The basic principle of photoexcitation is a homogeneous immune response. It is based on two particles surface coated antigens or antibodies, forming immune complexes in the liquid phase, drawing the two particles closer together. Under the excitation of laser, the transfer of ionic oxygen between particles occurs, and then high-energy red light is generated, and the photon number is converted into the target molecule concentration through a single photon counter and mathematical fitting. When the sample does not contain target molecules, immune complexes cannot be formed between the two particles, the distance between the two particles exceeds the transmission range of the ionic oxygen, the ionic oxygen is rapidly quenched in a liquid phase, and no high-energy-level red light is generated during detection. The following problems are difficult to solve at present when using a photoexcitation luminescence method to detect HIV: (1) The preparation process is complex, especially the microsphere preparation and modification process is too complex; (2) high false positive rate.
Therefore, development of an HIV detection kit that can be mass produced, has low cost, acceptable quality, and stable performance, and can meet both sensitivity requirements and linearity requirements is highly desirable.
Disclosure of Invention
The invention aims to solve the technical problem of providing a human immunodeficiency virus antibody detection kit aiming at the defects of the prior art. When the kit is applied to homogeneous chemiluminescence analysis detection, the inventor of the application unexpectedly finds that the kit has ultrahigh sensitivity and wide detection range.
Based on this, the present invention provides in one aspect a human immunodeficiency virus antibody detection kit comprising a reagent R1, said reagent R1 comprising a first buffer solution and, suspended therein, acceptor particles capable of generating a chemiluminescent signal upon reactive oxygen species, said acceptor particles comprising a carrier having an interior filled with a luminescent composition, the surface of said carrier having HIV antigen bound thereto; the receptor particles have a ZETA potential value of not higher than 0mV and not lower than-15 mV, preferably not higher than-5 mV and not lower than-10 mV.
A sugar content of not less than 40 μg per milligram mass of the acceptor particle;
the sugar content in the acceptor particle per milligram mass is not less than 50. Mu.g, preferably not less than 60. Mu.g.
The content of polysaccharide in the first buffer solution is 0.01 to 1wt%, preferably 0.05 to 0.5wt%.
The receptor particle size distribution variation coefficient C.V value of the R1 reagent in the kit is not lower than 5% and not higher than 20%. The polysaccharide is selected from the group consisting of carbohydrates containing three or more unmodified or modified monosaccharide units; preferably selected from the group consisting of dextran, starch, glycogen, inulin, levan, mannan, agarose, galactan, carboxydextran and aminodextran; more preferably selected from the group consisting of dextran, starch, glycogen and polyribose, most preferably dextran or dextran derivatives.
The sugar content was measured using the anthrone method.
The kit further comprises a reagent R2, the reagent R2 comprising a biotin-labeled HIV antigen.
The kit also includes an Anti-HIV sample diluent.
The kit also includes an Anti-HIV negative control.
The kit also includes an Anti-HIV-1 positive control containing calf serum and HIV-1 positive serum.
The kit also includes an Anti-HIV-2 positive control containing calf serum and HIV-2 polyclonal antibodies.
The kit also includes an Anti-HIV weak positive control containing calf serum and HIV-1 positive serum.
The beneficial effects of the invention are as follows: the potential value of receptor particles ZETA of the R1 reagent in the kit is not higher than 0mV and not lower than-15 mV; the R1 reagent is more stable, can meet the commercial requirement of mass production, has ultrahigh sensitivity and has a very wide detection range. The invention controls the sugar content in the receptor particles, reduces the influence of sugar on detection signals, improves the detection precision and reduces the production cost. At the same time, the method comprises the steps of,
drawings
FIG. 1 is a Gaussian distribution diagram of the receptor particles prepared in example 1.
FIG. 2 is a graph of a standard for sugar content measurement.
Detailed Description
In order that the invention may be readily understood, the invention will be described in detail. Before the present invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. The practice of the invention is not limited to the following examples, but is intended to be within the scope of the invention in any form and/or modification thereof.
Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless defined otherwise, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.
I terminology
The term "active oxygen" as used herein refers to a substance which is composed of oxygen in the body or in the natural environment, contains oxygen and is active in nature, and is mainly an excited oxygen molecule, including an electron reduction product of oxygen, superoxide anion (O 2 Hydrogen peroxide (H), a two-electron reduction product 2 O 2 ) Hydroxyl radical (OH) of three-electron reduction product, nitric oxide and singlet oxygen (1O) 2 ) Etc.
The term "acceptor particle" as used herein refers to a particle comprising a compound capable of reacting with reactive oxygen species to produce a detectable signal. The donor particles are induced to activate by energy or an active compound and release active oxygen in a high energy state which is captured by the acceptor particles in close proximity, thereby transferring energy to activate the acceptor particles. In some embodiments of the invention, the acceptor particle comprises a luminescent composition and a carrier, the luminescent composition being filled in the carrier and/or coated on the surface of the carrier. The "carrier" according to the invention is selected from the group consisting of tapes, sheets, rods, tubes, wells, microtiter plates, beads, particles and microspheres, which may be microspheres or microparticles well known to the person skilled in the art, which may be of any size, which may be organic or inorganic, which may be expandable or non-expandable, which may be porous or non-porous, which has any density, but preferably has a density close to that of water, preferably floats in water, and is composed of transparent, partially transparent or opaque materials. The carrier may or may not be charged and when charged is preferably negatively charged. The carrier may be latex particles or other particles containing organic or inorganic polymers, lipid bilayers such as liposomes, phospholipid vesicles, oil droplets, silica particles, metal sols, cells and microcrystalline dyes.
In the present invention, the "light-emitting composition", i.e., a compound called a label, may undergo a chemical reaction to cause light emission, such as by being converted into another compound formed in an electronically excited state. The excited state may be a singlet state or a triplet excited state. The excited state may relax to the ground state to emit light directly or by transferring excitation energy to an emission energy acceptor, thereby restoring itself to the ground state. In this process, the energy acceptor particles will be transitioned to an excited state to emit light.
The term "C.V value of the particle size distribution coefficient of variation" as used herein refers to the coefficient of variation of the particle size in the Gaussian distribution in the result of the detection by the nanoparticle analyzer. The calculation formula of the variation coefficient is as follows: C.V values = (standard deviation SD/Mean) x 100%. The standard deviation (Standard Deviation, SD), also called standard deviation, describes the average of the distances (from mean deviation) of the individual data from the average, which is the square root after the sum of the squares of the deviations, denoted sigma. The standard deviation is the arithmetic square root of the variance. The standard deviation reflects the degree of dispersion of a data set, and the smaller the standard deviation, the less the values deviate from the average and vice versa. The standard deviation sigma is the distance from the inflection point (0.607 times the peak height) on the normal distribution curve to the perpendicular line of the peak height and the time axis, i.e., half the distance between the two inflection points on the normal distribution curve. The half height peak width (Wh/2) refers to the peak width at half the peak height, wh/2=2.355 σ. The intercept at the base line is called the peak width or base line width, w=4σ or w=1.699 Wh/2, by making tangents to the inflection points on both sides of the normal distribution curve.
The term "ZETA potential" as used herein refers to the potential of the acceptor particle in an aqueous (pH equal to about 7) dispersion. The ZETA potential (Zetapotential) of a microsphere refers to the potential of the microsphere at the shear plane; i.e. between the continuous phase and the fluid-stabilizing layer attached to the microspheresPotential difference. Because the disperse particle surface has charges to attract surrounding opposite sign ions, the opposite sign ions are distributed in a diffusion state at a two-phase interface to form diffusionElectric double layer. The electric double layer can be divided into two parts according to the Stern electric double layer theory, namely a Stern layer and a Stern layerDiffusion layer. The Stern layer is defined as a planar layer composed of ionic (IHP or OHP) charge centers adsorbed on the electrode surface, the planar layer being relatively far from the interfaceThe potential at a point in the fluid is called the stem potential. Stabilizing layer [ ]Stationarylayer) (including a Stern layerSliding surfacePartial diffusion layer within a slip plane) and within a diffusion layerDispersing medium(dispersion medium) the interface upon relative movement is a sliding surface (slip plane), where the potential at a point in the fluid remote from the interface is referred to as the ZETA potential or electrokinetic potential (ZETA potential), i.e., the ZETA potential is between the continuous phase and the fluid stabilizing layer attached to the dispersed particlesPotential difference. It can pass throughElectric power Dynamic phenomenonAnd (5) directly measuring. The current method for measuring ZETA potential mainly comprisesElectrophoresis methodElectroosmosis, potentiodynamic and ultrasound, of which electrophoresis is the most widely used. The microsphere ZETA potential in the embodiment of the invention can be detected by the following method: NICOMP 380Z 3000 uses Doppler Electrophoresis (ELS) to determine the ZETA potential value. The ZETA potential value is obtained mainly by measuring the electrophoretic migration rate of charged particles in a suspension. The stability of the colloid was judged by measuring the ZETA potential of the microspheres.
The term "test sample" as used herein refers to a mixture to be tested that contains or is suspected of containing a target molecule to be tested. Samples to be tested that may be used in the present invention include body fluids such as blood (which may be anticoagulated blood as is commonly found in collected blood samples), plasma, serum, urine, semen, saliva, cell cultures, tissue extracts, and the like. Other types of samples to be tested include solvents, seawater, industrial water samples, food samples, environmental samples such as soil or water, plant material, eukaryotic cells, bacteria, plasmids, viruses, fungi, and cells from prokaryotes. The sample to be measured can be diluted with a diluent as required before use. For example, in order to avoid the HOOK effect, the sample to be tested may be diluted with a diluent before on-machine testing and then tested on a testing instrument.
The term "target molecule to be detected" as used herein refers to a substance in a sample to be detected during detection. One or more substances having a specific binding affinity for the target molecule to be detected may be used to detect the target molecule. The target molecule to be tested may be a protein, peptide, antibody or hapten which can be conjugated to an antibody. The target molecule to be detected may be a nucleic acid or oligonucleotide that binds to a complementary nucleic acid or oligonucleotide. The target molecule to be tested may be any other substance that can form a specific binding pair member. Examples of other typical target molecules to be measured include: drugs such as steroids, hormones, proteins, glycoproteins, mucins, nucleoproteins, phosphoproteins, drugs of abuse, vitamins, antibacterial agents, antifungal agents, antiviral agents, purines, antitumor agents, amphetamines, heteronitrogen compounds, nucleic acids and prostaglandins, and metabolites of any of these drugs; pesticides and metabolites thereof; and a recipient. Analytes also include cells, viruses, bacteria, and fungi.
The term "antibody" as used herein is used in its broadest sense and includes antibodies of any isotype, antibody fragments that retain specific binding to an antigen, including but not limited to Fab, fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, bispecific antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. In any desired case, the antibody may be further conjugated to other moieties, such as a member of a specific binding pair member, e.g., biotin or avidin (a member of a biotin-avidin specific binding pair member), and the like.
The term "antigen" as used herein refers to a substance that stimulates the body to produce an immune response and binds to antibodies and sensitized lymphocytes, which are the products of the immune response, in vivo and in vitro, resulting in an immune effect.
The term "binding" as used herein refers to the direct association between two molecules due to interactions such as covalent, electrostatic, hydrophobic, ionic and/or hydrogen bonding, including but not limited to interactions such as salt and water bridges.
The term "specific binding" as used herein refers to the mutual recognition and selective binding reaction between two substances, and from a steric perspective, corresponds to the conformational correspondence between the corresponding reactants. Under the technical ideas disclosed in the present invention, the detection method of the specific binding reaction includes, but is not limited to: a diabody sandwich method, a competition method, a neutralization competition method, an indirect method or a capture method.
Detailed description of the preferred embodiments
The present invention will be described in more detail with reference to examples.
In one aspect, the invention provides a kit for detecting an antibody of human immunodeficiency virus, comprising a reagent R1, wherein the reagent R1 comprises a first buffer solution and acceptor particles suspended therein and capable of generating a chemiluminescent signal by reacting with active oxygen, and the kit is characterized in that: the receptor particles comprise a carrier, the interior of the carrier is filled with a luminous composition, and the surface of the carrier is bonded with HIV antigen; the sugar content in the acceptor particle per milligram by mass is not less than 40. Mu.g, and at the same time, the ZETA potential of the acceptor particle is not more than 0mV and not less than-15 mV.
The carrier surface is coated with polysaccharide molecules and the HIV antigen is indirectly bound to the surface of the receptor particles by chemical bonding to the polysaccharide molecules.
When the variation coefficient of the particle size distribution of the receptor particles is more than or equal to 5% and not more than 20%, the kit containing the receptor particles has the capability of detecting low-concentration samples and better HOOK resistance. The receptor particles may have a value of 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% of the variation coefficient C.V of the particle size distribution in the reagent R1.
Preferably, the sugar content in the acceptor particle per milligram mass is not less than 50. Mu.g, preferably not less than 60. Mu.g.
Preferably, the polysaccharide content of the first buffer solution is 0.01 to 1wt%, preferably 0.05 to 0.5wt%.
In the above embodiments, the polysaccharide is selected from the group consisting of carbohydrates containing three or more unmodified or modified monosaccharide units; preferably selected from the group consisting of dextran, starch, glycogen, inulin, levan, mannan, agarose, galactan, carboxydextran and aminodextran; more preferably selected from the group consisting of dextran, starch, glycogen and polyribose, most preferably dextran or dextran derivatives.
In the above examples, the sugar content was measured by the anthrone method.
The receptor particles have a ZETA potential of not more than 0mV and not less than-15 mV, preferably not more than-5 mV and not less than-10 mV.
The kit further comprises a reagent R2, the reagent R2 comprising a biotin-labeled HIV antigen.
The kit also includes an Anti-HIV sample diluent.
The kit also includes an Anti-HIV negative control.
The kit also includes an Anti-HIV-1 positive control containing calf serum and HIV-1 positive serum.
The kit also includes an Anti-HIV-2 positive control containing calf serum and HIV-2 polyclonal antibodies.
The kit also includes an Anti-HIV weak positive control containing calf serum and HIV-1 positive serum.
The kit comprises a plurality of reagent strips, wherein each reagent strip is provided with a plurality of reagent hole slots for containing reagents, and at least one reagent hole slot is used for containing the reagents R1.
The invention also provides a use method of the human immunodeficiency virus antibody detection kit, which comprises the following steps of diluting a sample to be detected with a diluent, adding a reagent R1 and a reagent R2 into the diluent, adding a photosensitive reagent into the diluent, irradiating with laser, calculating photon quantity, and judging whether the sample to be detected is HIV negative or HIV positive according to the photon quantity.
The preferred method for using the human immunodeficiency virus antibody detection kit comprises the following steps:
step 1: diluting a sample to be tested by using an Anti-HIV sample diluent, and fully and uniformly mixing;
step 2: adding diluted sample, anti-HIV negative control, anti-HIV-1 positive control, anti-HIV-2 positive control and Anti-HIV weak positive control into the reaction well;
step 3: sequentially adding a reagent R1, a reagent R2 and a photosensitive reagent into the reaction hole;
step 4: and (3) placing the sample into a light-activated chemiluminescence detector, irradiating the reaction hole by laser, calculating the photon quantity emitted by the reaction hole, and judging HIV positive or HIV negative according to the photon quantity.
The invention also provides application of the human immunodeficiency virus antibody detection kit to a chemiluminescent analyzer.
III. Examples
Example 1 preparation of acceptor particle a
1.1 preparation of polystyrene latex microspheres
1) A100 mL three-necked flask was prepared, 40mmol of styrene, 5mmol of acrolein and 10mL of water were added thereto, and after stirring for 10 minutes, N was introduced 2 30min；
2) 0.11g of ammonium persulfate and 0.2g of sodium chloride were weighed and dissolved in 40mL of water to prepare an aqueous solution. Adding the aqueous solution into the reaction system of the step 1), and continuously introducing N 2 30min；
3) Heating the reaction system to 70 ℃ and reacting for 15h;
4) The emulsion after completion of the reaction was cooled to room temperature and filtered through a suitable filter cloth. Washing the obtained emulsion by centrifugal sedimentation with deionized water until the conductivity of the supernatant fluid at the beginning of centrifugation is close to that of the deionized water, diluting with water, and preserving in an emulsion form;
5) The average particle diameter of the Gaussian distribution of the latex microsphere particle diameter at this time was 202.2nm as measured by a nanoparticle sizer.
1.2 landfill Process of luminescent compositions
1) A25 mL round bottom flask was prepared and charged with 0.1g of a dimethylthiophene derivative and 0.1g of europium (III) complex (MTTA-EU) 3+ ) 10mL of 95% ethanol is magnetically stirred, the temperature of the water bath is raised to 70 ℃ to obtain a complex solution;
2) Preparing a 100mL three-neck flask, adding 10mL 95% ethanol, 10mL water and 10mL of aldehyde polystyrene latex microspheres with the concentration of 10% obtained in the step 1.1, magnetically stirring, and heating to 70 ℃ in a water bath;
3) Slowly dripping the complex solution in the step 1) into the three-neck flask in the step 2), stopping stirring after reacting for 2 hours at 70 ℃, and naturally cooling;
4) Centrifuging the emulsion for 1h,30000G, and removing supernatant after centrifuging to obtain aldehyde polystyrene microsphere filled with luminous composition.
1.3 surface coating of receptor particles with polysaccharide
1) 50mg of aminodextran solid was taken in a 20mL round bottom flask, 5mL of 50 mm/ph=10 carbonate buffer was added, and the solution was stirred at 30 ℃ in the absence of light;
2) Taking 100mg of prepared aldehyde polystyrene microspheres filled with the luminous composition, adding the prepared aldehyde polystyrene microspheres into an aminodextran solution, and stirring for 2 hours;
3) 10mg of sodium borohydride was dissolved in 0.5ml of 50 mM/pH=10 carbonate buffer, and then added dropwise to the above reaction solution, followed by reaction at 30℃overnight in the absence of light;
4) After the reaction mixture was centrifuged at 30000G, the supernatant was discarded, and 50 mM/ph=10 carbonate buffer was added for ultrasonic dispersion. After repeating the centrifugal washing three times, the volume is fixed by 50 mM/pH=10 carbonate buffer solution to make the final concentration of the solution be 20mg/mL;
5) 100mg of aldehyde dextran solid was taken in a 20mL round bottom flask, 5mL of 50 mm/ph=10 carbonate buffer was added, and stirred at 30 ℃ in the absence of light for dissolution;
6) Adding the microsphere into an aldehyde dextran solution, and stirring for 2 hours;
7) 15mg of sodium borohydride was dissolved in 0.5ml of 50 mM/pH=10 carbonate buffer, and then added dropwise to the above reaction solution, followed by reaction at 30℃overnight in the absence of light;
8) After the reaction mixture was centrifuged at 30000G, the supernatant was discarded, and 50 mM/ph=10 carbonate buffer was added for ultrasonic dispersion. After repeating the centrifugation washing three times, the final concentration was set to 20mg/mL by constant volume with 50 mM/pH=10 carbonate buffer.
9) The average particle size of the Gaussian distribution of the particle sizes of the microspheres at this time was 241.6nm as measured by a nanoparticle sizer (as shown in fig. 1).
1.4 Coupling process of HIV antigen and receptor particles
1) HIV antigen i was dialyzed to 50mM CB buffer at ph=9.0, measuring a concentration of 1mg/mL.
2) Into a 2mL centrifuge tube, 0.5mL of the receptor particles obtained in step 2.3 and 0.5mL of HIV antigen I were added, and after mixing, 100. Mu.L of 10mg/mL NaBH was added 4 Solution (50 mM CB buffer)(activator), 2-8deg.C for 4h.
3) After completion of the reaction, 0.5mL of 100mg/mL BSA solution (50 mM CB buffer) (blocking agent) was added, and the mixture was reacted at 2-8℃for 2 hours.
4) After the reaction, the mixture was centrifuged for 45min at 30000G, the supernatant was discarded after centrifugation, and resuspended in 50mM MES buffer. The centrifugation and washing were repeated four times, and diluted to a final concentration of 100. Mu.g/mL to obtain a receptor particle solution coupled with HBsAg antigen I, to obtain receptor reagent A.
5) The average particle diameter of the Gaussian distribution of the particle diameters of the microspheres at this time was 247.1nm as measured by a nanoparticle sizer.
Example 2 detection of sugar content of microspheres
1) Pretreatment of microsphere samples:
taking acceptor reagent A containing 1mg of acceptor microsphere a in example 1, centrifuging 20000G for 40min, pouring out supernatant, performing ultrasonic dispersion by using purified water, repeating the centrifugal dispersion for three times, and then using purified water to fix the volume to 1mg/mL.
2) Preparing a glucose standard solution:
1mg/mL of glucose stock solution was prepared as a standard solution of 0mg/mL, 0.025mg/mL, 0.05mg/mL, 0.075mg/mL, 0.10mg/mL, 0.15mg/mL with purified water.
3) Preparation of anthrone solution: 2mg/mL of the solution (stable at room temperature for 24h, ready-to-use) was prepared with 80% sulfuric acid solution.
4) To the centrifuge tube, 0.1mL of glucose standard solution and sample to be tested with each concentration were added, and each tube was filled with 1mL of anthrone test solution.
5) Incubation was carried out at 85℃for 30min.
6) The sample reaction tube 15000G is centrifuged for 40min, and the pipette tip sucks clarified liquid from the bottom of the tube to measure absorbance, so as to avoid sucking out suspended matters on the upper part.
7) The temperature was returned to room temperature and absorbance at 620nm was measured (preferably within 2 h).
8) The relationship between the sugar content concentration and the absorbance of the standard solution is shown in table 1, the sugar content concentration of the standard solution is taken as an X value, the absorbance is taken as a Y value, and a linear regression is performed to obtain a sugar content measurement standard curve shown in fig. 2, and the sugar content concentration of the sample to be measured is measured based on the sugar content measurement standard curve.
TABLE 1
| Sequence number | Concentration mg/mL | Absorbance A | Absorbance B | Absorbance mean |
| 1 | 0 | 0.0008 | 0.0008 | 0.0008 |
| 2 | 0.025 | 0.0880 | 0.0916 | 0.0898 |
| 3 | 0.05 | 0.1547 | 0.1611 | 0.1579 |
| 4 | 0.075 | 0.2375 | 0.2471 | 0.2423 |
| 5 | 0.1 | 0.3190 | 0.332 | 0.3255 |
| 6 | 0.15 | 0.4855 | 0.5053 | 0.4954 |
Detection result:
the absorbance of the solution of the receptor microsphere a in example 1 was measured to be 0.1984, and the sugar content of the receptor microsphere a was 60.5. Mu.g/mg based on a sugar content measurement standard curve.
Example 3 preparation of HIV detection kit and performance verification thereof
The kit consists of a reagent R1 (prepared from the example 1), a reagent R2 (biotin-labeled HIV antigen), and a photosensitive reagent R3 containing donor particles, an Anti-HIV negative control, an Anti-HIV-1 positive control, an Anti-HIV-2 positive control, an Anti-HIV weak positive control, and an Anti-HIV sample diluent. The components are respectively prepared, the reagent R1 and the reagent R2 are combined into an Anti-HIV kit, and the negative control, the positive control, the weak positive control and the sample diluent are respectively and independently split-packed and then assembled into the kit. The preparation process is summarized as follows:
the preparation of reagent R1 (receptor particle coating HIV antigen): the receptor particles prepared in example 1 were prepared by adding a first buffer solution at a concentration of 50. Mu.g/mL.
Reagent R2 (biotin-labeled HIV antigen): the treated HIV antigen and biotin are mixed uniformly according to a certain concentration and proportion to react to form a connector, and after dialysis, a certain amount of buffer solution is added to prepare the HIV antigen.
third-HIV negative control: anti-HIV qualitative reference dilutions.
Anti-HIV-1 positive control: diluting HIV-1 positive serum with Anti-HIV qualitative reference diluent.
Fifthly, anti-HIV-2 positive control: the HIV-2 polyclonal antibody is diluted by using Anti-HIV qualitative reference diluent.
Sixth, anti-HIV weak positive control: diluting HIV-1 positive serum with Anti-HIV qualitative reference diluent.
The kit can carry out qualitative detection on HIV (1+2 type) antibody, and the detection is carried out by using matched Anti-HIV weak positive control, anti-HIV negative control, anti-HIV-1 positive control and Anti-HIV-2 positive control, wherein the Anti-HIV weak positive control is added with 2 holes, the Anti-HIV negative control, the Anti-HIV-1 positive control and the Anti-HIV-2 positive control are respectively added with 1 hole. The reagent needs to equilibrate to ambient temperature before use.
Step 1: diluting the sample to be tested with Anti-HIV sample diluent for 11 times, and fully mixing (e.g. adding 10ul sample into 100 ul sample diluent);
step 2: adding 25 mu L of diluted sample, anti-HIV negative control, anti-HIV-1 positive control, anti-HIV-2 positive control and Anti-HIV weak positive control into the reaction well;
step 3: sequentially adding 25 mu L of a reagent R1, 25 mu L of a reagent R2 and 25 mu L of a reagent R3 into the reaction well;
step 4: placing a LiCA500 full-automatic photo-excitation chemiluminescence detector produced by Boyang biotechnology (Shanghai) limited company into the device, and automatically operating the device, wherein the method comprises the following specific steps:
A. vibration type
B.37℃incubation for 15min
C. 175 mu L of photosensitive agent is automatically added
Incubation at 37℃for 10min
E. Laser irradiates the microwells and calculates the amount of photons emitted from each well
F. And calculating an S/CO value (the ratio of the optical signal value of the sample to be detected to the weak positive Anti-HIV reference optical signal) by software, and judging the negative and positive.
[ method for judging validity of kit ]
For each test, weak positive Anti-HIV control, negative Anti-HIV control, positive Anti-HIV-1 control, and positive Anti-HIV-2 control are added
The S/CO value of the Anti-HIV negative control should be less than 0.6, the S/CO value of the Anti-HIV-1 positive control should be more than or equal to 3, the S/CO value of the Anti-HIV-2 positive control should be more than or equal to 3, if the result is abnormal, the test result is not credible and needs to be repeated.
[ method for judging detection results of kit ]
S: the optical signal value of the sample to be measured;
CO: anti-HIV weak positive control light signal value (CUT OFF reference value);
the software automatically calculates the S/CO value, the sample to be tested is judged to be negative when the S/CO is less than 1, and the sample to be tested is judged to be positive when the S/CO is more than or equal to 1.
The kit of this example has the following good properties as tested:
(1) Negative reference compliance rate: detecting by using national reference, wherein the coincidence rate of 20 HIV antibody negative reference is not less than 18;
(2) Positive reference compliance rate: the national reference is used for verification, 18 HIV-1 type antibody positive references cannot be false negative, and RLUP12 is more than or equal to RLUP11;2 HIV-2 type antibody positive samples should not have false negative;
(3) Sensitivity: performing detection by using a national reference, wherein 1 part of matrix serum in 6 parts of sensitivity reference serum is negative, and at least 3 parts of diluted serum are positive;
(4) Precision: verification with national reference C.V +.15% (n=10);
(5) Clinical sensitivity, clinical specificity: among the tests of 511 serum samples judged to be positive clinically, 511 samples were detected to be positive, the clinical sensitivity reached 100%, and among the tests of 581 serum samples judged to be negative clinically, 580 samples were detected to be negative, the clinical specificity reached 99.83%.
Example 4 influence of sugar content on the detection results
The experimental steps are as follows:
1. selecting 10 tumor patient interference samples, and verifying the interference samples as HIV anergy samples by a verification reagent; and 20 positive HIV samples verified by a validation reagent;
2. preparing receptor particles with different sugar contents, coupling HIV antigens, preparing the receptor particles into a concentration of 20 mug/mL, and testing signal values after 30 samples are reacted;
3. after the S/CO value of each sample is calculated, the detection result is positive when the S/CO value is more than or equal to 1, and the detection result is negative otherwise;
4. the false positive result is the false positive result when the positive result appears in the tumor interference sample, and the missed detection is the negative result appears in the HIV positive sample.
The test results are shown in table 2 below:
TABLE 2
When the sugar content of the receptor particles is not less than 40 mug, the sensitivity is high, the detection capability of the low-value sample is high, and the false positive sample is less disturbed.
Example 5: determination of ZETA potential of receptor particles in receptor reagent
The invention adopts a ZETA potential detection method: NICOMP 380Z 3000 uses Doppler Electrophoresis (ELS) to determine the ZETA potential value. The ZETA potential value is obtained mainly by measuring the electrophoretic migration rate of charged particles in a suspension. The stability of the colloid was judged by measuring the ZETA potential of the microspheres. The effect of the ZETA potential of a particle is mainly that the particle surface is charged.
The method for measuring the ZETA potential comprises the following steps:
1) Acceptor reagents of different ZETA potentials were prepared in the manner described in example 1 (acceptor reagents of different ZETA potentials were obtained by adjusting the addition amounts of blocking agent and activator, etc. during the coupling of HIV antigen to acceptor particles), wherein the sugar content per mg of the acceptor particles was not less than 40. Mu.g in each acceptor reagent, as measured by the anthrone method.
2) Samples were prepared and the different acceptor reagents prepared in step 1) were diluted to 10 μg/mL in deionized water.
3) The NICOMP 380Z 3000 instrument is calibrated by a standard product, and then the ZETA potential is measured. The results are shown in Table 3.
4) Preparation of HOOK sample for HIV project
Defining the lowest detection limit as 0.5NCU Kang Che Si tan quality control product detection capability judgment, and when the test signal of a sample of a certain experimental group is just larger than the CO signal, namely RLU (Cx) > RLU (C0), determining that the test result is positive. A HOOK sample is defined as a sample with a higher concentration of the analyte than the concentration of the antigen-antibody in the reagent, the test result has a risk of low false nature, and the test result is S/co=rlu (Cx)/RLU (C0).
The experimental procedure was as follows:
(1) Taking a 0.5NCU Kang Che Si tan quality control sample as a detection limit sample, and judging the detection capability difference through the S/CO value and the yin-yang property;
(2) Diluting the HOOK sample into a series of samples by 2 times of gradient, and then testing;
5) Different Zeta potential receptor particles are selected to prepare reagents, the HOOK sample of the HIV project is detected, and the deviation between the theoretical value and the original factor is calculated under different dilution factors.
TABLE 3 Table 3
Analysis of results: the experimental group shows that when the HOOK sample of the HIV project is measured, the original multiple measurement result is lower when the Zeta potential (mV) is lower than-15 mV, the theoretical value deviation is larger, and the HOOK phenomenon is easy to occur; and above-15 mV, this phenomenon does not occur, indicating that the HOOK resistance is stronger at this time.
Example 6: detection of HIV antibody levels in samples of normal humans and patients suspected of being infected with HIV virus
Clinical verification of HIV agent (receptor particles of example 1):
1. randomly selecting 100 normal physical examination samples, 50 tumor patient samples and 50 suspected HIV virus infection patients;
2. the receptor particles with the sugar content of 60 mug are coupled with HIV antigen to prepare a concentration of 20 mug/mL, and the concentration is reacted with 200 samples to test signal values;
3. after the S/CO value of each sample is calculated, the detection result is positive when the S/CO value is more than or equal to 1, and the detection result is negative otherwise;
the test results are shown in Table 5 below:
TABLE 5
Analysis of results: the coincidence rate of 150 negative samples is 100%, and no false positive exists; the coincidence rate of 50 positive samples is 100%, no missed detection exists, and the clinical diagnosis requirement of HIV reagent is met.
Claims (18)
1. A human immunodeficiency virus antibody detection kit comprising a reagent R1, said reagent R1 comprising a first buffer solution and suspended therein receptor particles capable of generating chemiluminescent signals upon interaction with reactive oxygen species, characterized in that: the receptor particles comprise a carrier, the interior of the carrier is filled with a luminous composition, and the surface of the carrier is bonded with HIV antigen; the surface of the carrier is coated with polysaccharide molecules, and the sugar content in each milligram of the receptor particles is not less than 40 mug; the receptor particles have a ZETA potential value of not greater than 0mV and not less than-15 mV.
2. The kit of claim 1, wherein: the receptor particles have a ZETA potential value of not more than-5 mV and not less than-10 mV.
3. The kit of claim 1, wherein: the HIV antigen is indirectly bound to the surface of the receptor particle by chemical bonding to the polysaccharide molecule.
4. The kit of claim 1, wherein: the sugar content in the acceptor particle per milligram mass is not less than 50 mug.
5. The kit of claim 4, wherein: the sugar content in the acceptor particle per milligram mass is not less than 60 mug.
6. The kit of any one of claims 1-5, wherein: the first buffer solution contains polysaccharide, and the content of the polysaccharide in the first buffer solution is 0.01-1 wt%.
7. The kit of claim 6, wherein: the content of polysaccharide in the first buffer solution is 0.05-0.5 wt%.
8. The kit of claim 1 or 6, wherein: the polysaccharide is selected from the group consisting of carbohydrates containing three or more unmodified or modified monosaccharide units.
9. The kit of claim 8, wherein: the polysaccharide is at least one selected from dextran, starch, glycogen, inulin, levan, mannan, agarose, galactan, polyribose, and dextran derivatives.
10. The kit of claim 9, wherein: the polysaccharide is selected from carboxydextran and/or aminodextran.
11. The kit of claim 1, wherein: the sugar content was measured using the anthrone method.
12. The kit of any one of claims 1-5, wherein: the receptor particle size distribution variation coefficient C.V value of the R1 reagent in the kit is not lower than 5% and not higher than 20%.
13. The kit of claim 1, further comprising a reagent R2, wherein the reagent R2 comprises a biotin-labeled HIV antigen.
14. The kit of claim 1, further comprising an Anti-HIV sample diluent.
15. The kit of claim 1, further comprising one or more of an Anti-HIV negative control, an Anti-HIV-1 positive control, an Anti-HIV-2 positive control, and an Anti-HIV weak positive control.
16. A method of using the human immunodeficiency virus antibody detection kit according to any one of claims 1 to 15, comprising the steps of diluting a sample to be detected with a diluent, adding a reagent R1 and a reagent R2 thereto, adding a photosensitive reagent thereto, irradiating with a laser, calculating the photon quantity, and judging whether the sample to be detected is HIV negative or HIV positive based on the photon quantity.
17. A method of using the human immunodeficiency virus antibody detection kit of claim 16, comprising the steps of:
step 1: diluting a sample to be tested by using an Anti-HIV sample diluent, and fully and uniformly mixing;
step 2: adding diluted sample, anti-HIV negative control, anti-HIV-1 positive control, anti-HIV-2 positive control and Anti-HIV weak positive control into the reaction well;
step 3: sequentially adding a reagent R1, a reagent R2 and a photosensitive reagent into the reaction hole;
step 4: and (3) placing the sample into a light-activated chemiluminescence detector, irradiating the reaction hole by laser, calculating the photon quantity emitted by the reaction hole, and judging HIV positive or HIV negative according to the photon quantity.
18. Use of a human immunodeficiency virus antibody detection kit according to any one of claims 1-15 in a chemiluminescent analyzer.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2019114122740 | 2019-12-31 | ||
| CN201911412274 | 2019-12-31 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113125714A CN113125714A (en) | 2021-07-16 |
| CN113125714B true CN113125714B (en) | 2023-06-16 |
Family
ID=76772360
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011637955.XA Active CN113125714B (en) | 2019-12-31 | 2020-12-31 | Human immunodeficiency virus antibody detection kit and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113125714B (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2140661A1 (en) * | 1992-07-20 | 1994-02-03 | Sharat Singh | Novel chemiluminescent compounds and methods of use |
| US5340716A (en) * | 1991-06-20 | 1994-08-23 | Snytex (U.S.A.) Inc. | Assay method utilizing photoactivated chemiluminescent label |
| WO1999042838A1 (en) * | 1998-02-18 | 1999-08-26 | Dade Behring Inc. | Chemiluminescent compositions for use in detection of multiple analytes |
| WO2007113337A1 (en) * | 2006-04-06 | 2007-10-11 | Tibotec Pharmaceuticals Ltd. | A homogeneous time resolved fluorescence based test system |
| WO2014047222A2 (en) * | 2012-09-19 | 2014-03-27 | Abbvie Biotherapeutics Inc. | Methods for identifying antibodies with reduced immunogenicity |
| US8916341B1 (en) * | 2013-11-04 | 2014-12-23 | Allied Innovative Systems, Llc | Methods for improving analyte detection using photochemical reactions |
| CN109725153A (en) * | 2017-10-26 | 2019-05-07 | 北京科美生物技术有限公司 | Homogeneous phase immunoassay method and application thereof |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2287006T3 (en) * | 2000-03-06 | 2007-12-16 | Dade Behring Marburg Gmbh | VEHICLES COVERED WITH POLYSACARIDS, ITS PREPARATION AND USE. |
| CN104090101A (en) * | 2014-07-21 | 2014-10-08 | 威海威高生物科技有限公司 | Human immunodeficiency virus (HIV) antibody detection kit and preparation method thereof |
| CN106546732A (en) * | 2015-09-17 | 2017-03-29 | 深圳迈瑞生物医疗电子股份有限公司 | Human immunodeficiency virus antigen and antibody combined detection kit and application thereof and detection method |
| CN110161230A (en) * | 2018-02-11 | 2019-08-23 | 博阳生物科技（上海）有限公司 | Quickly homogeneous immune reagent kit, preparation method, detection method and the device of detection Procalcitonin |
| CN109988333B (en) * | 2019-04-04 | 2023-04-07 | 成都爱兴生物科技有限公司 | Polystyrene microsphere |
| CN110133252A (en) * | 2019-05-10 | 2019-08-16 | 湖北民族大学 | For detecting kit and detection method and its application of carcinomebryonic antigen |
-
2020
- 2020-12-31 CN CN202011637955.XA patent/CN113125714B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5340716A (en) * | 1991-06-20 | 1994-08-23 | Snytex (U.S.A.) Inc. | Assay method utilizing photoactivated chemiluminescent label |
| CA2140661A1 (en) * | 1992-07-20 | 1994-02-03 | Sharat Singh | Novel chemiluminescent compounds and methods of use |
| WO1994002486A1 (en) * | 1992-07-20 | 1994-02-03 | Syntex (U.S.A.) Inc. | Novel chemiluminescent compounds and methods of use |
| WO1999042838A1 (en) * | 1998-02-18 | 1999-08-26 | Dade Behring Inc. | Chemiluminescent compositions for use in detection of multiple analytes |
| WO2007113337A1 (en) * | 2006-04-06 | 2007-10-11 | Tibotec Pharmaceuticals Ltd. | A homogeneous time resolved fluorescence based test system |
| WO2014047222A2 (en) * | 2012-09-19 | 2014-03-27 | Abbvie Biotherapeutics Inc. | Methods for identifying antibodies with reduced immunogenicity |
| US8916341B1 (en) * | 2013-11-04 | 2014-12-23 | Allied Innovative Systems, Llc | Methods for improving analyte detection using photochemical reactions |
| CN109725153A (en) * | 2017-10-26 | 2019-05-07 | 北京科美生物技术有限公司 | Homogeneous phase immunoassay method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Deceased tissue donor serology and molecular testing for HIV, hepatitis B and hepatitis C viruses: a lack of cadaveric validated tests;Thayssa Neiva da Fonseca Victer 等;Cell Tissue Bank;17(4);全文 * |
| 均相光激化学发光免疫分析技术研究进展;颜露;赵晓航;许杨;;生命科学(09);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113125714A (en) | 2021-07-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP3839485B1 (en) | Microsphere composition for chemiluminescence analysis and use thereof | |
| CN116754757A (en) | Donor particle for homogeneous chemiluminescence analysis and application thereof | |
| CN116500020A (en) | Chemiluminescence analysis method and application thereof | |
| CN113125715B (en) | Human immunodeficiency virus antibody detection kit and application thereof | |
| EP3508849B1 (en) | Antibody measurement method using antigen-carrying insoluble carrier particles on which antigen is immobilized by different methods, and reagent for antibody measurement | |
| CN113125714B (en) | Human immunodeficiency virus antibody detection kit and application thereof | |
| CN113125711B (en) | Acceptor reagent and application thereof | |
| CN112240930B (en) | Homogeneous chemiluminescence analysis method and application thereof | |
| CN113125709A (en) | Donor reagent and application thereof | |
| CN112240928B (en) | Homogeneous chemiluminescence analysis method and application thereof | |
| CN112114131A (en) | Homogeneous phase chemiluminescence detection method and application thereof | |
| CN112114130A (en) | Receptor reagent for homogeneous phase chemiluminescence detection and application thereof | |
| CN113391064A (en) | Receptor reagent for detecting novel coronavirus neutralizing antibody and application thereof | |
| CN114966007A (en) | Microsphere composition for homogeneous phase chemiluminescence detection | |
| CN113125419B (en) | Donor reagent and application thereof | |
| CN113125703B (en) | Myoglobin homogeneous detection kit and application thereof | |
| CN113125705B (en) | Myoglobin homogeneous detection kit and application thereof | |
| CN113125721B (en) | Homogeneous detection kit for creatine kinase isozymes and application thereof | |
| CN113125701B (en) | Homogeneous chemiluminescence detection kit and application thereof | |
| CN113125700B (en) | Homogeneous detection kit for creatine kinase isozymes and application thereof | |
| CN113125417A (en) | Receptor reagent and application thereof | |
| CN113125732B (en) | Homogeneous detection kit for interleukin 6 and application thereof | |
| CN113125730B (en) | Homogeneous detection kit for interleukin 6 and application thereof | |
| CN113125698A (en) | Receptor reagent and application thereof | |
| CN113125702A (en) | Homogeneous phase chemiluminescence detection kit and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information |
Address after: 201210 the third and fifth floors of Building 1, No. 88, Cailun Road, Pudong New Area, Shanghai Applicant after: Kemei Boyang diagnostic technology (Shanghai) Co.,Ltd. Applicant after: Kemei Diagnostic Technology Co.,Ltd. Address before: 201210 the third and fifth floors of Building 1, No. 88, Cailun Road, Pudong New Area, Shanghai Applicant before: BEYOND DIAGNOSTICS (SHANGHAI) Co.,Ltd. Applicant before: Kemei Diagnostic Technology Co.,Ltd. |
|
| CB02 | Change of applicant information | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |